Project Summary/Abstract: Thyroid cancer is the most common malignancy of endocrine tissues and most patients require lifelong surveillance with diagnostic thyroid-stimulating hormone (TSH)- stimulated radio-iodine imaging as well as remnant or metastatic ablation with radioiodine therapy. Other imaging techniques including ultrasound and positron-emission tomography have also gained increasing importance as follow-up surveillance methods. However, these other methods are not intrinsically specific for thyroid tissue and there remains a need for novel, specific thyroid imaging methods, especially for tumors increasingly recognized by thyroglobulin production but with poor radio-iodide uptake due to absence or loss of the relatively labile sodium-iodide symporter. The PI, who was both co-inventor and co-developer with Genzyme of wild type recombinant human TSH, currently approved for diagnostic imaging of thyroid cancer, now proposes to develop novel 123I or 125I-labeled high affinity TSH analogs for imaging of thyroid cancer through the specific and relatively stable TSH receptor. Our previous structure- function studies resulted in the discovery of the first analogs of TSH and other glycoprotein hormones with major increases in receptor binding affinity and bioactivity ("superactive analogs"). In the current proposal, we plan to produce, purify and characterize large amounts of such novel TSH analogs for commercial development as imaging agents. We propose in Aim 1 to develop stable cell lines producing high levels of candidate superactive analogs and produce quantities sufficient for initial imaging studies. This Aim will consist of construction of dicistronic vectors with amplifiable markers and mutated subunit cDNAs, followed by amplification, production, purification and characterization of unpurified analogs. Selected TSH analogs will then be labeled with 123I or 125I by the lactoperoxidase method and characterized by various physicochemical methods as well as their binding affinity to the TSH receptor. Aim 2 will consist of gamma camera imaging of both primary and metastatic thyroid tumors with 123I-TSH in a novel transgenic murine model of thyroid cancer. These mice express human RET/PTC3 solely in the thyroid and develop thyroid hyperplasia, solid tumor variants of papillary carcinoma and metastatic cancer. In addition, quantitative uptake studies of 125I-TSH analogs will be performed in various organs and in all sites of primary and metastatic thyroid tumors. This novel labeled TSH imaging method may later be extended to novel therapeutic approaches for thyroid cancer including TSH receptor-mediated delivery of therapeutic agents such as radionuclides or toxins as well as TSH-receptor localizing gene therapy approaches. Project Narrative: Thyroid cancer is the most common malignancy of endocrine tissues and most patients require lifelong surveillance with diagnostic thyroid-stimulating hormone (TSH)-stimulated radio-iodine imaging to detect recurrent tumor. The PI has developed a much improved form of TSH and proposes a completely new method of thyroid cancer imaging that should greatly improve early detection and lead to improved cancer treatment and survival. [unreadable] [unreadable] [unreadable]